2441_C11

269

11

Food Pasteurization and Sterilization with High Pressure

Alberto Bertucco

University of Padova, Italy

Sara Spilimbergo

University of Trento, Italy

CONTENTS

11.1 Introduction ..................................................................................................27011.2 Ultrahigh Hydrostatic Pressure Treatment (UHHP)....................................271

11.2.1 Process and Equipment Fundamentals ............................................27111.2.2 State of the Art of UHHP ................................................................272

11.2.2.1 Microorganisms ................................................................27211.2.2.2 Enzymes............................................................................27411.2.2.3 Microbial Inactivation Mechanism...................................274

11.2.3 Commercial Application of UHHP .................................................27811.3 Dense CO

2

Treatment (DCO

2

).....................................................................28011.3.1 Process and Equipment Fundamentals ............................................28111.3.2 State of the Art of DCO

2

.................................................................28111.3.2.1 Microorganisms ................................................................28111.3.2.2 Enzymes............................................................................28411.3.2.3 Microbial Inactivation Mechanisms .................................285

11.3.3 Commercial Application of DCO

2

...................................................28711.4 Potentials of High-Pressure Technologies and Conclusions .......................288References..............................................................................................................289

2441_C011.fm Page 269 Thursday, July 20, 2006 10:13 AM

© 2007 by Taylor & Francis Group, LLC

270

Functional Food Ingredients and Nutraceuticals

11.1 INTRODUCTION

Manufacturers of food products are currently under increasing and stringentdemands to control their production process. The entire course of manufacturingstarting from raw materials to the final packing is closely monitored and whereverpossible inspected as well. This is happening essentially because of the growingdemand for high-quality products and for energy-saving and safer productionprocesses. Consumers and the business class today prefer fresh or mildly processedand user-friendly food products. Typically, such products are low in sugar, salt,and fat, and contain as few preservatives as possible. However, these factors offeran excellent breeding ground for microorganisms. Thus, finding the right methodfor extending the shelf-life for these highly perishable products without affectingtheir quality is indeed an open challenge. Microbiological stabilization of a productis not concerned with the removal of all existing microorganisms but to reducethe number of undesirable ones below a specific critical value during the shelf-life of the product.

On the other hand, the use of food preservatives and additives is viewed withmore and more concern by consumers, as shown by the increase in restrictivemeasures appearing in the food regulations of industrialized countries. The appre-hension about these substances is associated with the concerns for heat treatmentsthat are still the most widely used procedure in food industry to microbiologicallystabilize foodstuff. Thermal pasteurization (up to 80°C) and sterilization (up to120°C) are successful treatments in eliminating the degradation effects of enzymesand microorganisms, but they may also decrease food quality by causing alterationsin the taste and sensory attributes of food products. As a consequence, in the future,the food industry is expected to turn toward new and alternative technologies forextending shelf life while retaining all original nutritional properties in the endproducts. In this regard, systems based on different bactericidal agents, such asmicrowave, electrical conductivity, pressure gradient, and so forth, seem to offernew possibilities. They include high pressure (both hydrostatic and with CO

2

), pulsedelectric field,

1

ohmic heating,

2,3

pulsed x-ray,

4

ultraviolet light,

5

ultrasound,

6

filtra-tion,

7

microwave and radio frequency processing,

8

and oscillating magnetic fields.

9

Although these technologies have been studied for more than one century, at thepresent time none of them is ready to market.

In particular, as far as high-pressure treatments are concerned, for many yearsresearchers have investigated the effect of high hydrostatic pressure both on micro-organism viability and on foodstuff properties.

10,11

The main result has been thatsuch a technique can be successfully applied at room temperature, however, the highplant costs will restrict its development for large-scale production. On the otherhand, the use of supercritical CO

2

seems to be a more feasible approach, since itallows operations at milder temperature (close to ambient) and pressure (70–300bars).

12

However, this new technique has been proposed only recently, thus noindustrial application is ready to enter the large-scale market yet.

This chapter will describe and discuss the current knowledge in the pasteuriza-tion and sterilization of foodstuff by means of UltraHigh Hydrostatic Pressure(UHHP) method and Dense CO

2

(DCO

2

) method. The most significant achievements




271

will be summarized, including fundamental concepts, main applications in bothsimple and complex solutions, and results on inactivation of different microorgan-isms and enzymes. At the end of the chapter the current knowledge on the differenthypotheses of bacterial inactivation mechanisms involved will be discussed andevaluated and some conclusions about the two high-pressure techniques will bedrawn.

11.2 ULTRAHIGH HYDROSTATIC PRESSURE TREATMENT (UHHP)

Pressure is one of nature’s fundamental forces that is known to have unique effectson the thermodynamics of substances. Likewise, the preservation action of highpressure on food substances has been acknowledged in the food science communityfor over 100 years.

13

The isostatic nature of pressure results in no shear stress withinthe food and thus the food’s shape is not destroyed. However, the thermodynamicconditions achieved under UHHP treatment are sufficiently hostile to living bacteriathat they are inactivated or irreparably damaged. In this way potential foodbornepathogens can be destroyed and food spoilage organisms can also be greatly reduced.Since this process is typically carried out at temperatures not far from ambient, thefood quality can be retained.

11.2.1 P

ROCESS

AND

E

QUIPMENT

F

UNDAMENTALS

The use of UHHP treatment of food is carried out in batch mode. A process schematicis reported in Figure 11.1. The UHHP equipment essentially consists of a relativelylarge pressure-resistant vessel and a high-pressure generating system. A boosterpump and an intensifier pump are used to reach the required pressure. Food packagedin waterproof sealed elastic containers ranging from bottles to bags are placed intothe vessel, which is then filled with water (or water-glycol solution) and pressurizedto typically 500–600 MPa for a time period usually between 2 and 3 minutes.

The pressure is applied by a direct or indirect compression technique but themore widespread method is the indirect one, with a pump compressing the liquidfrom the medium-pressure tank to the cell (see Figure 11.1), until the desired pressurevalue is reached. The temperature control is ensured by a simple electrical resistanceif only heating is required. Otherwise a heating/cooling jacket, or a heat exchangerinside the cell, can be applied. The form of the container is designed to minimizethe dead space inside the autoclave. It is important for reaching a high volumetricefficiency in order to reduce the cost per unit. Under routine operation, foods arenow processed at pressures up to 600 MPa. Single plant production rates of 40million lb/yr are already in operation. Production costs typically range from over 6to about 4 cents/lb.

14



272


11.2.2 S

TATE

OF

THE

A

RT

OF

UHHP

11.2.2.1 Microorganisms

Relevant studies about the effect of high pressure on different kind of microbes andfood substrates will be briefly discussed in this section. Early experiments, dated inthe late 19th and early 20th centuries, showed that short treatments with an operatingpressure of a few thousand bars were able to reduce the microbial activity by manyorders of magnitude.

13

In 1899 it was observed that pressurized milk would remainfresh and unspoiled for a longer time than untreated milk; also microbes containedin vegetables and fruits could be inactivated if they underwent a high-pressuretreatment for a few minutes.

15,16

Many later studies also showed that short treatments with an operating pressure

of a few thousand bars were able to reduce the vegetative forms of microbial activityby various orders of magnitude.

17

For instance, Timson and Short in 1965 started asystematic investigation to test the resistance of bacterial spores, and to try toinactivate them completely, that is, to get sterilization. These authors studied thebehavior of spores under UHHP treatment with longer processing times at constantpressure in a range of temperatures between –25°C and 95°C.

18

With the works ofGould and coworkers,

19,20

the existence of optimal hydrostatic pressure to inactivatespores was demonstrated. After much research on various bacterial forms, these

FIGURE 11.1

Schematic of typical UHHP apparatus.

Pre

ssu

re b

oo

ster

Pump

High-pressure cell

Pressure medium tank

P




273

authors confirmed their hypothesis about the induction of spore germination by theaction of pressure. More recently, Ludwig’s group investigated the behavior of sporesunder different conditions of UHHP

21

and introduced the cycle-type treatment thatproved to be more efficient than the double-level treatment.

22,23

In recent years the effect of high hydrostatic pressure on the survival of differentkinds of microorganism in various food substrates under different conditions wasaddressed

24–33

and reviewed.

26,27

Generally, the most recent results confirm the find-ings of early studies that food can be pasteurized under high pressure (400–600MPa) and low to moderate temperatures (up to 60°C) but these products requirerefrigeration during storage and distribution to ensure microbiological stability. Thespecific operating conditions depend on the history of the bacterial cultures, that is,the type and growth phase of the test microorganisms considered.

25

Bacterial sporeswere demonstrated to require more severe pressure treatments, in combination withother preservation techniques, principally heat treatment, to achieve inactivation.

31,34

Recently, high hydrostatic pressure inactivation of vegetative microorganism,

aerobic, anaerobic spores in pork Marengo (a low acidic particulate food product)was studied by Moerman,

35

who showed that

Saccharomyces cerevisiae

and theGram-negative bacteria

Pseudomonas fluorescens

and

Escherichia coli

are morepressure sensitive than

cocci Enterococcus faecalis

and

Staphylococcus aureus

(Gram-positive): their inactivation at room temperature was successful only withpressures as high as 600 MPa, whereas Gram-negative bacteria were more easilykilled at pressure of about 400 MPa. The UHHP method was also applied to solidsubstrates, in particular air-dried alfalfa seeds inoculated with

E. coli

and

L. mono-cytogenes

. These samples were subjected to different pressure conditions (from275–575 MPa for 2 min and 475 MPa for 2–8 min, at 40°C).

36

It was shown that amaximum reduction of 2 log can be obtained, and that treated seeds took a longertime to germinate compared to the untreated seed. The effect of high-pressureprocessing on the safety, quality, and shelf life of ready-to-eat meats (low-fat pas-trami, Strasburg beef, export sausage, and Cajun beef) was investigated at 600 MPaand 20°C for 3 min.

10

After processing, samples were stored at 4°C for 98 days.After storage their counts of aerobic and anaerobic mesophilies, lactic acid bacteria,

Listeria

spp., staphylococci,

Brochothrix thermosphacta

, coliforms, yeasts, andmolds were undetectable or at low levels. Furthermore, sensory analyses revealedno difference in consumer acceptability and no sensory quality degradation. Aninteresting paper concerning the effect of high-pressure-induced inactivation of

Listeria innocua

in buffer frozen suspension was published by Luscher et al.;

37

acycle pressure treatment above 200 MPa resulted in inactivation of about 3 log,probably due to the mechanical stress associated with phase transition of ice intoits different polymorphs. The effect of various pressure levels (50–600 MPa) andholding times on color and microbiological quality of bovine muscle was alsoinvestigated.

38

The reported experiments, carried out at 10°C and pressure higherthan 300 MPa, induced modifications of meat color parameters. Pandey et al.

39

studied the effect of high pressure treatment (250–400 MPa) for various holdingtimes (0–80 min) at 3°C and 2°C on raw milk with high count of indigenousmicroflora. It was found that higher pressures, longer holding times, and lowertemperatures resulted in larger destruction of microorganisms.



274


Finally, we quote an interesting paper by Linton et al.

40

who found that UHHPtreatment (at 20°C and 300–600 MPa) readily inactivated psychotropic bacteria,

coliforms,

and

pseudomonas

in different types of shellfish such as mussels, prawns,scallops, and oyster.

Table 11.1 is a compilation of interesting UHHP applications to food that canbe found in the literature from the year 2000 on, with indication of the substrate,the type of microorganism, the pressure conditions, and the maximum inactivationratio achieved.

11.2.2.2 Enzymes

Food quality deterioration is caused by a wide range of phenomena, includingphysical conditions and both chemical and biochemical reactions. As far as enzy-matic reactions are concerned, the effect of pressure on protein structure and func-tionality can vary dramatically depending on the magnitude of the pressure, thereaction mechanism, and the overall balance of forces responsible for maintainingthe protein structure.

41

To date, it is difficult to establish precisely the general effect of hydrostatic

pressure on different enzymes in various food substrates and environment. Pressurehas always been recognized as a potential denaturant of proteins, but examples ofpressure-induced stabilization have also been reported. For instance, both polyphenoloxidases (PPO) and peroxidases (POD) which play an important role in food quality,as they influence the visual appearance, flavor and health-promoting properties, areknown to be pressure-stable enzymes.

42

This stability depends on plant source andtype of product (e.g., whole fruit, rather than puree or juice).

The global effect of pressure on enzymes is quite complex and can be explainedin terms of individual molecular interactions within proteins, including hydropho-bic, electrostatic, and van der Waals interactions.

41

The information available onthis topic is scarce and often contradictory; thus it is still impossible to discussexhaustively the effect of UHHP on most enzymes catalyzing chemical reactionswhich influence quality and degree of deterioration of foodstuff. Table 11.2 providea summary of recent studies on the effect of pressure on enzymatic reactionsrelated to food, with indication of enzyme type, treatment conditions, and substrateutilized.

11.2.2.3 Microbial Inactivation Mechanism

Microorganisms are inactivated when they are exposed to factors that substantiallyalter their cellular structure or physiological functions. Structural damage includesDNA strand breakage, cell membrane rupture, and mechanical damage to cell enve-lope. The reason for the effect of high pressure on microorganisms is not completelyclear. It is well known that pressures in the range of 20–180 MPa delay microbialgrowth and tend to inhibit protein synthesis, while at pressures higher than 180 MPainactivation causes the loss of viability. Lethal UHHP treatment disrupts membraneintegrity and denatures many proteins. Another fundamental requirement for thesurvival and viability of microorganisms is the regulation of the cytoplasmatic pH




275

TABLE 11.1Applications of UHHP on Microbial Cells in Simple and Complex Solutions

ReferenceTreatment

RegimeTemperature

(°C)MaximumReduction

InoculatedMicroorganism

Solution/Substrate

35 400 MPa/30 min

20–50 1.31 log

Bacillus subtilis

Pork marengo

0.14 log

Bacillus stearothermophilus

0.74 log

Clostridium sporogenes

0.21 log

Clostridium tyrobutyricum

0.69 log

Clostridium saccharolyticum

1.63 log

Enterococcus faecalis

1.79 log

Staphylococcus aureus

3.35 log

Escherichia coli

6.49* log

Pseudomonas fluorescens

3.51* log

Saccharomyces cerevisiae

36 575 MPa/2 min 475 MPa/2–8 min

40 1.4 log2.0 log

Escherichia coliListeria monocytogenes

Alfalfa seeds

82 300 MPa/5 min400 MPa/1 min700–800 MPa/5 min

900 MPa/1 min

20 4* log

Salmonella typhimuriumEscherichia colYersinia enterocoliticaVibrio parahaemolyticusBacillus cereusStaphylococcus aureusListeria monocytogenes

0.1% Buered peptone water (pH 7.4)

10 600 MPa/180 s 20 4* log

Anaerobic mesophilesLactic acid bacteriaListeria spp.Staphylococci Brochothrix thermosphacta

Coliforms yeast and molds

Low-fat pastrami Strasburg beef Export sausage Cajun beef

37 200 MPa Subzero temperature

3 log

Listeria innocua

Buffer solution

83 >75 MPa/30 min

37

Escherichia coli

Desoxycholate Agar (DESO)

84 200 MPa/8 min

Escherichia coli

85 150–250 MPa Not inactivated at 250 MPa

Listeria monocytogenes

Chilled cold-smoked salmon

* means total inactivation



276


TABLE 11.1 (CONTINUED)Applications of UHHP on Microbial Cells in Simple and Complex Solutions

ReferenceTreatment

RegimeTemperature



Solution/Substrate

86 350–550 MPa 30–45 7* log

Listeria monocytogenesStaphylococcus aureus;Gram-negative:Escherichia coliSalmonella enteritidis

UHT 1% low fat milk

87 350 MPa/20 min

50 5 log

Alicyclobacillus acidoterrestris

Model system (BAM broth) and orange, apple, and tomato juices

38 50–600MPa/20–300 s

10 2.5 Total flora Bovine muscle

39 250–400MPa/0–80 min

2–31

Escherichia coli

Raw milk

40 300–600MPa/2 min

20 96% Psychrotrophic bacteriaColiformsPseudomonas

Shellfish, mussels, prawns, scallops, oysters

81 50–400MPa/15 min

25 4 log Total aerobic mesophilic and psychrotrophic bacteria

Tomato puree + natural additives (citric acid and sodium chloride)

47 400MPa/10 min

20 9* log


pH 5,6 citrate buffer

88 450MPa/15 min

40 99.97% Porcine blood plasma

89 400MPa/20 min

Room temperature

2 log


ACES buffer (N-(2-aceta-mido)-2-aminoethane-sulfonic acid)

90 500MPa/60 s

Room temperature

4–5 log Natural flora Green beans

91 300–600 MPa Milk and diary products

92 400–500MPa/5–30 min

10–40 Red blood cells fraction from porcine blood





277


ReferenceTreatment

RegimeTemperature



Solution/Substrate

75 300, 800 MPa Room temperature

L. plantarumEscherichia coli

93 300MPa/15 min

5–50 Lactic acid bacteriaBaird Parker flora

Pseudomonas

sp.Enterobacteria

Sliced cooked ham and ground pork patties

94 200–600 MPa Lactobacillii harmful to beer

Beer

95 50–400 MPa/1–60 min

20–80 6* log3.67 log2.27 log

Bacillus subtilisBacillus stearothermophilus

Streptococcus faecalis

Meat batters

96 1000 MPa/15 min

20 Not inactivated at 1000 MPa

B. cereus

Fruit and vegetable products

34 400 MPa/25 min

8–30 0.4 log

Listeria monocytogenesBacillus cereusPseudomonas fluorescens

Ultrahigh temperature milk

97 400 MPa/10 min600 MPa/10 min


Citrate buffer and phosphate buffer

98 200 MPa/12h 25 4,7 log Psychrophilic bacteria Tilapia fillets99 545 MPa

Escherichia coliListeria monocytogenes enteroxigenic

Staphylococcus aures

Tomato-based salsa

100 600 MPa/5min Yeast and lactic acid bacteria

Beer

101 400 MPa/three 5-min cycle

7 2 log Microbial load Chilled hake (

Merluccius capensis

) 102 400 MPa/5 min 20 7 log

Escherichia coli

Milkcheese

103 100–500MPa/15 min

4-25-50

Escherichia coliPseudomonas fluorescensListeria innocuaStaphylococcus aureusLactobacillus helveticus

Ringer solution and ovine milk




278


which plays an important role in secondary transport of several compounds; anirreversible internal pH decrease during UHHP treatment (200–300 MPa) probablypromotes denaturation of proteins required for pH homeostasis.

43

Another effect tobe taken into account is a phase transition of the cytoplasmatic membrane from thephysiological liquid-crystalline to the gel phase, which can induce leakage of sodiumand calcium ions and increase membrane permeability.

44

The main structural andfunctional changes in microorganisms at different pressures are summarized inFigure 11.2.

It is worth noting that these microbial inactivation hypotheses were establishedrecently, thanks to the possibility of exploiting new analytical methods which candetermine structural and physiological cell modifications and loss of cytoplasmaticcontent by quantitatively and qualitatively estimating viable cells. This field ofresearch was started because the traditional methods of analysis were not able toassess the physiological state of damaged cells after UHHP treatment. Furthermore,classic culture techniques were widely recognized to underestimate the number oftruly viable bacteria, especially when cells had been damaged by physical treatment.Fluorescent staining, detection by microscopy, flow cytometry, and differential scan-ning calorimetry are presently the most widely used techniques.

37,45–47

11.2.3 C

OMMERCIAL

A

PPLICATION

OF UHHP

In the last 15 years the food industry has been successful in marketing a number ofUHHP treated products, which meet evolving regulatory concerns for greater foodsafety and a growing consumer demand for higher-quality and convenient (i.e.,ready-to-eat) foods. Commercialization is being achieved thanks to collaborationbetween food scientists, microbiologists, and high-pressure equipment providers.

Products and processes using UHHP are now available. In 1990 the Meidi−yaFood Company of Japan, introduced the first UHHP-pasteurized products, namelystrawberry, kiwi, and apple jams. From 1993 the range of this kind of products hasbecome larger and larger, and a variety of jams, juices, sauces, milk-desserts, fruitjellies, raw beef, and fish have been commercially produced. These products are


ReferenceTreatment

RegimeTemperature



Solution/Substrate

104 400 MPa/10 min continuous pressure and pulsed pressure in two 5-min steps

7 5 log H2S-producing microorganisms

Lactic acid bacteriaBrochothrix thermosphacta

Coliforms

Oysters



Food Pasteurization and Sterilization with High Pressure 279

TABLE 11.2Applications of UHHP on Enzymes in Food Substrates

ReferenceTreatment

RegimeTemperature

(°C) EnzymesSolution/Substrate

105 >300 MPa/ 20 min 9 Proteolytic enzymes: Cathepsin B-likeCathepsin B+L-like Calpains

Cold-smoked salmon

106 400, 600, 800MPa/5, 10, 15 min

18÷22 Beta-glucosidase Peroxidase Polyphenoloxidase

Red raspberry and strawberry

107 Fruits and vegetables108 Pectin

methylesterase PME

Carrots

109 Oxidase enzymes Muscadine grape juice

110 Proteolytic enzymes Octopus arm muscle111 300 450 MPa/

15 minBeta-lactoglobulin

112 400 MPa/10 min 7 Polyphenoloxidase Oysters74 300 MPa

113 100–400 MPa Proteins Alphalactalbumin Beta-lactoglobulin

Milk

114 0–450 MPa/15–30 min

7-40-75-100 Microbial load and autolytic activity

Octopus muscles

115 207–310 MPa/0–2 min

Lipase Pacific oysters

116 395–445 MPa/8–11 min

70 Carrot juice

117 0–800 MPa Tomato pectin methylesterase

polygalacturonase

Tomato

118 50 MPa/72 h Protein Cheese119 Rhizomucor miehei

lipase120 Up to 700 MPa 20÷65 Lactoperoxidase Bovine milk and acid

whey121 600–900 MPa Nonflavonoid

phenolicsGrape juice

122 300–400 MPa,30–120 min

4.4 log3.09 log

Pectin methyl esterase

Orange juice

÷ in the range– separates single set points



280 Functional Food Ingredients and Nutraceuticals

packed in plastic bags and can be stored at 4°C for two months if sealed, or for oneweek after opening. At the present time, fruit, vegetables, shellfish, meat, and otherproducts are in commercial UHHP production.48 In Europe, at least two well-established UHHP pasteurized products are worth mentioning, fruit juice in Franceand ham in Spain, while in Mexico companies are treating avocado puree for theU.S. market at a processing pressure of 700 MPa.17 For the sake of completeness,it must be mentioned that UHHP can be applied at the industrial scale to reduce themicrobial activity, as well as to modify the consistency of foodstuffs; active researchhas been developed to obtain UHHP precooked foods such as meat and rice-basedproducts.49

11.3 DENSE CO2 TREATMENT (DCO2)

The antimicrobial effect of CO2 under pressure (or dense CO2) was discovered inthe second half of last century, but specific research began only about 20 years ago.For instance, in the book on dense gas extraction by Quirin et al.,50 part of a chapteris dedicated to “sterilization” by dense gases, showing considerable reduction (i.e.,5 to 8 log) of microbial counts after appropriate CO2 pressure application at roomtemperature. Soon afterward, a systematic investigation was started to exploit thiseffect in order to develop a new nonthermal pasteurization technology, suitable forapplications where processing temperatures close to ambient have to be used.

As detailed in the section “state of the art” below, DCO2 was found to be lethalto basically all forms of microorganisms to which it had been applied. In addition,

FIGURE 11.2 Main structural and functional changes in microorganisms as a function ofpressure (adapted from Ref. 29).

300

200

100

50

0.1

Irreversible protein denaturation

leakage of cell content

Membrane damage

signs of cell content leakage

Inhibition of protein synthesis

reduction in the number of ribosomes

Intracellular pH decrease

Reversible protein denaturation

compression of gas vacuoles

Threshold of lethality

Atmospheric pressure

Pressure (MPa)




it has been shown51 that the antimicrobial action can be exerted at pressures as lowas 7 MPa, that is, much lower than the ones required by UHHP treatment. Thus,DCO2 qualifies as the best candidate to replace traditional thermal treatment, espe-cially when heat-labile components need to be preserved, as often happens withproducts related to food, pharmaceuticals, and cosmetics. A major limitation of theDCO2 technology is that direct contact between CO2 and the microorganism to bekilled must be ensured. Therefore, at present, pasteurization can be achieved suc-cessfully only when liquid substrates or slurries are processed; treating solid mate-rials by this technique remains problematic.

11.3.1 PROCESS AND EQUIPMENT FUNDAMENTALS

Treatment of liquid solutions and suspensions (i.e., pumpable substrates) by DCO2

is quite a simple task, which can be accomplished in many ways. The most efficienttype of equipment is a continuous contactor, where the feed to be pasteurized andthe CO2 stream can flow either cocurrently or countercurrently. A flow sheet of apilot unit is sketched in Figure 11.3. The plant basically consists of a CO2 surgetank, two high-pressure pumps (one for CO2, the other for the substrate to bepasteurized), a mixer where the two streams are suitably contacted, a thermostaticallycontrolled holding tube to ensure the proper retention time at the desired temperature,a suitable depressurization system, and eventually a degasser section.

Energy requirements of this process are generally low, as liquids are easier topump than gases. The consumption of CO2 is quite low as well: in the case ofaqueous-based substrates the ratio between the CO2 and feed flow rates is nevermore than 1:20 on a weight basis, because the solubility of CO2 in these liquids isusually less than 0.05 (weight fraction). If desired, after depressurization, CO2 maybe recompressed and recycled back to the contactor, but this option increases sub-stantially the energy requirement. From our experience, the retention (holding) timeto get a 6-log count reduction depends on the type of microorganism, but is usuallyin the range of 5 to 30 min, and can be further reduced by a suitable design of theholding tube.52 So far, we have processed a number of fresh fruit juices (such asorange, apple, grape, and pear) in a unit like the one depicted in Figure 11.3, alwaysobtaining good results in terms of inactivation, provided that a temperature not lessthan 30°C–32°C was used.

11.3.2 STATE OF THE ART OF DCO2

11.3.2.1 Microorganisms

The possibility of using CO2 under pressure to inactivate microbes was initiallyaddressed by Fraser in the 1950s,53 who reported the disruption of bacteria cells byrapid release of CO2 gas from about 35 bars to ambient pressure. The first patent inthe field was obtained by Swift & Co. in 1969, who claimed that food productscould be sterilized by CO2 without degradation of their flavor at “superatmospheric”conditions and by exposing them to relatively low radiation dosages.54

Since 1985 many papers have reported on the bacteriostatic action of CO2 andon the growth and metabolism of different microorganisms, but it is from the paper




by Kamihira et al. in 198755 that the inhibitory effect of CO2 under pressure towardmicrobes started to be addressed systematically and quantitatively. The number ofpublications on this topic has increased over the years since 1990. Many authorshave reported experimental evidence on the effect of dense CO2 on different sub-strates and different kinds of microbes commonly present in foodstuff, both in theirvegetative and latent forms. A review on inactivation of bacteria by the DCO2 method

FIGURE 11.3 Schematic of DCO2 continuous equipment (adapted from Ref. 52); P1, P2,P3: pumps; PV: vacuum pump; A1, A2, B1, B2, D: tanks; F: filter; V1, V2, V3, V4, V5, V6:on-off valves; VNR: no-return valve; V-S: safety valve; VL: control valve; VP: samplingvalves; TI: temperature indicators; PI: pressure indicators; BT1, BT2: thermostatic baths; C:chiller; R: flow meter.

CO2

D

B1B2

A1 A2

V1 V2

P1

F C

P2

PI2

V3

VNR

V4

V-S

VP1

TI2

TI3

TI4

TI5

TI6

TI7

TCI

VP 2

VP5

VP4

VP3

VP6

VP6

PI3

P3

BT 1

BT2

VL

V-3

V5

PV

V6

PI4

PC

TI1

TI8

R

PI1




has been published recently,12 and it discusses research published in 83 articles andpatents, half of which were published from 1999 to 2003.

In the last two years several new articles have been published. They include anarticle by Liu et al.56 which describes a study on the influence of compression anddecompression rate, and the concerted effects of temperature, pressure, exposuretime, water content, and initial pH on the physiology of Absidia coerula and Sac-caromices cerevisiae. The paper by Erkmen57 deals with the mathematical analysisof high-pressure CO2 regarding the inactivating effect on S. cerevisiae at differenttemperatures and pressures. His study allowed the prediction of yeast inactivationwhen exposed to different CO2 operating conditions. Watanabe et al.58 comparedDCO2 treatment with other methods, in particular UHHP and thermal processing,for the inactivation of spores at different temperatures and exposure times. In thework by Furukawa et al.,59 the effect of CO2 on the germination of spores was studiedat 65 bars, 35°C, and 120 min.

In the past two years, several patents have also been issued, including the oneclaimed by Balaban,60 which deals with a continuous method to reduce microorgan-ism and enzyme activities in liquid beer and wine products, and the one claimed byPraxair Inc. (Burr Ridge, IL, USA), which validates a continuous DCO2 process asa nonthermal pasteurization technology of fruit juice from lab-scale to commercial-ization for a feed flow rate up to 120 L/min (Better than Fresh®). In this last patentthe presence of a CIP (Cleaning In Place) equipment is evaluated as a key designfeature, to assure frequent cleaning and sanitization of the apparatus.61

Very recently, more studies have been discussed at international symposiums onhigh pressure and supercritical CO2, which demonstrate the increasing interest inthis new mild pasteurization technology. Daiminger et al.62 presented a study on theefficiency of a continuous apparatus able to ensure 8-log count reduction of differentkinds of microbes, both inoculated in orange juice and naturally present in an activesludge. The effect of the main operating parameters (flow rate and pressure) wasdiscussed and an inactivation mechanism was proposed. Inactivation of Staphylo-coccus in liquid whole egg products by means of DCO2 in a batch stirred pilot devicewas presented by Van Ginneken et al.63 and patent application on this promisingprocess has been filed.64 Zhang and coworkers65 investigated the synergistic effectof DCO2 in conjunction with low levels of H2O2 to deactivate spores and found thatat least 4-log reduction can be reached at 40°C. On the inactivation mechanisms,disruption of the exospores, morphological changes, and release of dipicolinic acid(DPA) have been observed by means of TEM, SEM, and DPA fluorescence assay.

From these reports, it is apparent that DCO2 has been shown to be effective asa bactericidal agent on vegetative forms of microorganisms at a near-ambient tem-perature and a relatively low pressure. The experiments, performed using both simplesuspensions and complex substrates, resulted in inactivation levels sufficient to assurethe pasteurization of foodstuffs. However, it should be noted that the texture of somesolid products was disrupted and the color was changed after the DCO2 treatment,66

thus further work is needed in this respect. Mainly batch devices have been used asexperimental apparatus at the laboratory scale and different operating conditions ofpressure (55–300 bars) and temperature (10°C–50°C) have been tested. The presenceof a mixing system has been shown to be beneficial in terms of inactivation efficiency.




As far as latent forms are concerned, DCO2 alone is not suitable for practicalapplication in the food industry, unless higher temperatures (at least more than 60°C)are applied.67 In this case, some hurdle approach can be beneficial.25,68

11.3.2.2 Enzymes

As mentioned above, an irreversible pressure-induced denaturation of proteins gen-erally requires values of pressure greater than 300 MPa, much larger than the onesused in DCO2 treatment. A different hypothesis must therefore be proposed to justifythe inactivation of enzymes caused by DCO2. Apart from pressure, a number offactors can be taken into account when dealing with the influence of high-pressureCO2 on enzymes: pH of the medium, temperature, processing time, surface tensionat the gas-water interface, type of microbes, and nature of the substrate.

Current research results show that DCO2 treatment can either activate or inac-tivate enzymes.52 The interaction between dense CO2 and enzyme molecules isexpected to cause conformational changes that will result in either loss or increaseof activity. Recent reports dealing with the influence of high-pressure CO2 onenzymes are listed in Table 11.3 with indication of enzyme type, treatment condi-tions, and substrate utilized. It can be noticed that, despite the number of experi-mental data so far collected on enzyme activity changes after DCO2 treatment, nocorrelation nor definite enzymatic inactivation hypotheses are available in the liter-ature. For instance, the decrease of activity has been related to the loss of secondarystructures in enzyme molecules.69 Studies using gel electrophoresis showed that thereare differences in the isoelectric profiles and protein patterns between the untreatedand CO2-treated polyphenol oxidase (PPO), an enzyme causing undesirable brown-ing on the surface of fruits and vegetables. Spectropolarimetric analysis revealedthat CO2 treatment also changes the secondary structure of this enzyme.70 An enzymeH+-ATPase, located in the cell membrane, which is also important for the regulationof bacterial internal pH, was shown to maintain its initial specific activity in L.plantarum cell membrane even though viability of the cells was reduced by severallog counts after high-pressure treatment.71 In contrast, Wouters et al.72 observedstimulation of ATPase activity in the membrane vesicles when L. plantarum wasexposed to a hydrostatic treatment at 250 MPa for 80 min. The activity assessmentof several enzymes by using the APIZYM system from B. subtilis cells before andafter DCO2 treatment was exploited to demonstrate selective enzymatic inactivationas one of the probable cause of microbial inactivation.71 Recently, Habulin et al.73

observed that proteinase from Carica papaya latex showed improved stability at300 bars of CO2 pressure, when compared to ambient pressure, and that addition ofwater also increased the activity of this enzyme (optimum amount was between 0.5and 0.7 g/L).

On the so-called hurdle approach, two papers have recently been published. Bothof them deal with the combined effect of UHHP and DCO2 treatments on the activityof selective enzymes: Park and Lee74 observed that the residual activity of polyphe-noloxidases, lipoxygenase, and pectinmethylesterase were less than 11.3%, 8.8%,and 5.1%, respectively, after a combined treatment of DCO2 at 4.9 MPa and UHHPat 600 MPa. Corwin et al. studied the UHHP processing of CO2-added solutions (at




0.2% CO2 mole fraction). After treatment at 300÷800 MPa a synergistic effect onenzyme inactivation of pectin methyl-esterase and polyphenol-oxidase in L. plan-tarum and E.coli was found.75

11.3.2.3 Microbial Inactivation Mechanisms

If inactivation of microorganisms in a pumpable substrate can be achieved in asimple apparatus such as the one outlined in Figure 11.3, the reason for the abilityof DCO2 to inactivate microorganisms is not clear yet. Over the years, a number ofhypotheses have been proposed to explain the inactivation mechanisms, but to datenone appears completely satisfactory. For instance, it was demonstrated that, understandard operating conditions, cell death is not caused by explosive decompressionof CO2 upon pressure release.76 Also, the inactivation of key enzymes as a conse-quence of direct interaction with CO2 has not been sufficiently proven, and remainsa possible hypothesis. On the other hand, extraction of cell wall lipids by DCO2

based on the extraction capacity of CO2 as a supercritical fluid cannot be invokedat all as a reason for deactivation. Studies have shown that when the operatingpressure is as low as 7 MPa and the temperature is around 30°C, the solubility ofphospholipids in DCO2 is quite negligible. Thus, both intracellular acidification andmodification of the cell membrane properties remain the two main reasons for

TABLE 11.3Applications of DCO 2 Treatment on Enzymes in Simple and Complex Solutions

ReferenceTreatment

RegimeTemperature

(°C) Enzymes Solution/Substrate

123 31 MPa/10 min 30–60 Pectinesterase Orange juice69 25 MPa/30 min 35-45-55 Pectinesterase Valencia orange juice

124 Supercritical and gaseous liquid phase, pH 3 ÷ 6

25–50 Acid protease, alk. protease, papain, and glucoamylase

McIlvaine buffer

125 30MPa/30 min 35 Myoglobin Aqueous solution126 35, 2 MPa/15min;

62, 1 MPa/15 min40–55 Lipoxygenase, peroxidase 30% sucrose solution

127 30 MPa 35–50 Alpha-amilaseacid protease

Buffer solution

71 7 MPa/10 min 30 H+-ATPase, costitutive enzyme in L. plantarum

Saline solution

75 300–800 MPa(0.2% CO2)

25–30 Pectin methylesterase (PME), polyphenol oxidase (PPO)

128 Micro-bubble methods

Alpha-amilaseacid protease

Buffer solution

73 Batch, 300 bar 50 Proteinase from C. papaya




explaining cell deactivation. It is interesting that both reasons are associated withthe CO2 dissolution in the substrate.

To better explore this hypothesis, the data presented in Figure 11.4 may behelpful. Let us consider the acidification aspect first. When a suitable CO2 pressureis applied over a physiological aqueous solution where model microorganisms aresuspended, the pH of the solution is a strong function of pressure. It can be eithermeasured, for example, by online UV spectroscopy77 (results are shown in Figure11.4a), or calculated according to a suitable equation of state (SAFT)78 (Figure 11.5),so that curves such as those displayed can be obtained, depending on the systemtemperature. Clearly, when a pressure between 60 and 80 bars is reached, the pHvalue is lowered to about 3 by CO2 dissolution, but at higher CO2 pressures it cannotbe further reduced; this behavior is compatible and can be correlated with the trendof CO2 solubility in water.52,78

On the other hand, a saline solution at neutral pH can be completely pasteurizedby the DCO2 treatment carried out at 80 bars of pressure, so that it might beconcluded that such a pH decrease (down to about 3) is responsible for the deathof microorganisms. However, when a different aqueous solution such as orange juicewas considered for the same experiment, no pH effect could be detected uponapplication of CO2 pressure (as shown in Figure 11.4b), even though total deactiva-tion was achieved. This is an indication that solution (i.e., external) acidificationitself is not sufficient to explain the inactivation of microorganisms. As far asinteraction with the cell membrane is concerned, it was suggested that CO2 candissolve into phospholipids, even at pressures of 60–80 bars.79 If the CO2 concen-tration in the phospholipids becomes high enough, the physical properties of thecell membrane can be modified enough to allow the enzymes linked to the membranedouble layer to be released. On the other hand, the higher CO2 concentration in themembrane helps penetration of CO2 into the cytoplasm, with subsequent decreaseof intracellular pH value. As a consequence of internal acidification, other phenom-ena such as enzyme deactivation and salt precipitation are likely to occur.

It is noteworthy that both external acidification and modification of cell mem-brane properties are driven by the pressure-increased chemical potential of CO2 inthe gas phase contacting the aqueous solution. If sufficient time is allowed forequilibration after pressure is applied, thermodynamics dictates that the chemicalpotential has to be the same in all the phases involved, that is the liquid bulk, thecell membrane, and the cytoplasm, leading to the possible consequences discussedabove.

Experimental evidence of the proposed hypothesis is under current investigation.With respect to intracellular acidification, we have recently shown that it can bemeasured and correlated to the microbial inactivation level.79 As far as the propertychanges of cell membrane are concerned, preliminary results suggest that whencentrifuging an E.coli suspension after DCO2 treatment, significant and unexpectedkey enzyme activity can be measured in the liquid phase (supernatant), a clearindication of the enzyme release from the cell membrane itself.52




11.3.3 COMMERCIAL APPLICATION OF DCO2

Despite the huge research and development efforts performed in the last 20 years,at present no industrial applications of DCO2 to food pasteurization are known, eventhough several companies are actively working in this field. It appears that commer-cialization could be a matter of time. In this respect it is interesting to quote theopinion of Praxair, a company which is particularly active in developing a commer-cially sustainable DCO2 process,61 as reported in the proceedings of the recent Fifth

(a)

(b)FIGURE 11.4 Experimental extracellular pH profiles as a function of operating CO2 pressureat 35°C in water, pH = 7 at atmospheric condition (4a) and in orange juice, pH = 3.47 atatmospheric condition (4b).

35°C

2.80

2.90

3.00

3.10

3.20

3.30

3.40

Pressure (bar)

pH

Water

0 20 40 60 80 100 120 140 160 180

35°C

2.80

2.90

3.00

3.10

3.20

3.30

3.40

3.50

3.60

Pressure (bar)

pH

Juice

0 20 40 60 80 100 120 140 160 180




International Symposium on Supercritical Fluids (Orlando, FL, May 2005):80 “…The validation of the dense phase carbon dioxide process is two-fold. First, itdemonstrated a greater than 5-log reduction of the pertinent pathogens on juice usingthis process, to meet the juice HACCP guidelines set by FDA. As a result, thistechnology is now considered by the FDA as an alternative to thermal pasteurization.Second, product quality was validated by demonstrating the retention of physical,nutritional quality and sensory profile. …” Under these circumstances, it could beargued that the use of DCO2 as a nonthermal pasteurization treatment is going tobe reality within a short time.

11.4 POTENTIALS OF HIGH-PRESSURE TECHNOLOGIES AND CONCLUSIONS

From the results and discussion presented in this chapter, it can be concluded thathigh pressure provides efficient alternative processes for the pasteurization of food-stuffs when avoidance of thermal effects becomes an issue. It has been shown thatUHHP treatment is already a commercial technology, at least for niche products,whereas the DCO2 process needs more scale-up development before achievingindustrialization. On the other hand, it is clear that neither UHHP nor DCO2 alonecan be exploited as sterilization techniques, and that high-pressure processing hasto be performed in combination with other physical or chemical treatments81 in orderto kill all microorganisms, including latent forms. Of course, coupling differenttechniques could also be beneficial to achieve pasteurization, for instance by allowing

FIGURE 11.5 Calculated pH profiles as a function of CO2 pressure at different temperatures(taken from Ref. 78).

0 50 100 150 200 250 300 350 400 450 500

3.05

3.10

3.15

3.20

3.25

3.30

3.35

313.15 K

328.15 K333.15 K

pH

Pressure (bar)




the use of lower pressures and processing time, which would reduce the investmentsin pressure equipment25,27 and improve the process economics. It is also interestingthat in recent works the coupled effect of DCO2 and UHHP has been given specialattention.74,75

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